Fig. 2 |. Overview of CRISPRa/CRISPRi techniques.

Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa)/CRISPR interference (CRISPRi) provides a powerful set of tools to study gene function through precise and modulatory regulation of gene expression. CRISPRi techniques include empty nuclease-dead CRISPR-associated protein 9 (dCas9) (that is, with no effector domain), which is thought to attenuate gene expression via steric hindrance of RNA polymerase II (Pol II) binding to DNA¹⁰², and dCas9 tethered to the Krüppel-associated box (KRAB) domain, a potent transcriptional repressor. The CRISPRa techniques were developed using a fusion between dCas9 and VP64, a potent viral transcriptional activator. This toolbox continues to expand to include new methods to increase effector recruitment to a single gene and increase the magnitude of gene induction. Combining dCas9–VP64 with multiplexed guide RNAs (gRNAs) directed towards several locations along the same promoter synergistically activates transcription⁴⁵. In addition, dCas9 fused to multiple transcriptional effectors, including VP64, p65 and RTA (dCas9–VPR), substantially increases the magnitude of gene induction^12,13. Synergistic activation mediator (SAM) combines dCas9–VP64 with a gRNA scaffold that selectively recruits an RNA-binding protein tethered to the p65 and HSF1 activation domains. Last, SunTag and SunTag–p65–HSF1 (SPH) use a dCas9-bound protein scaffold that acts as a binding site for single-chain variable fragment (scFv)-fused transcriptional activators. The effector domains for SunTag and SPH are VP64 and p65–HSF1, respectively. The CRISPRa/CRISPRi methods are illustrated on a spectrum showing their estimated repression and activation of gene expression. TSS, transcription start site.